Clinical Chemistry 55:6
1061–1066 (2009)
Steroid Hormone Analysis by Tandem Mass Spectrometry
Steven J. Soldin1,2* and Offie P. Soldin1,3,4
BACKGROUND: New high-performance liquid chroma-
tography/tandem mass spectrometry (LC-MS/MS)
methods are among the most successful approaches
to improve specificity problems inherent in many
immunoassays.
CONTENT: We emphasize problems with immunoas-
says for the measurement of steroids and review the
emerging role of LC-MS/MS in the measurement of
clinically relevant steroids. The latest generation of tan-
dem mass spectrometers has superior limits of quanti-
fication, permitting omission of previously employed
derivatization steps. The measurement of steroid pro-
files in the diagnosis and treatment of congenital adre-
nal hyperplasia, adrenal insufficiency, chronic pelvic
pain and prostatitis, oncology (breast cancer), and ath-
letes has important new applications.
CONCLUSIONS: LC-MS/MS now affords the specificity,
imprecision, and limits of quantification necessary for
the reliable measurement of steroids in human fluids,
enhancing diagnostic capabilities, particularly when
steroid profiles are available.
© 2009 American Association for Clinical Chemistry
Steroid hormones are synthesized from cholesterol,
and many are of great clinical importance (1 ). Synthe-
sis occurs in the mitochondria and smooth endoplas-
mic reticulum of cells in the adrenal cortex, the gonads,
and the placenta (Fig. 1). The adrenal gland is com-
posed of the adrenal medulla and cortex. The latter is
divided into 3 anatomic zones: the zona glomerulosa,
which produces the mineralocorticoids such as cor-
ticosterone and aldosterone, and the zonae fascicu-
lata and reticularis, which together produce the
glucocorticoids (11-deoxycortisol, cortisol) and the ad-
1
Department of Medicine; and 2 Department of Pharmacology and General
Clinical Research Center; 3 Lombardi Department of Oncology, Comprehensive
Cancer Center; and 4 Department of Physiology and Biophysics, Georgetown
University, Washington, DC.
* Address correspondence to this author at: Bioanalytical Core Laboratory, Room
GM12A, Preclinical Science Building, Georgetown University, 3900 Reservoir
Road NW, Washington, DC 20007. E-mail sjs44@georgetown.edu.
Received October 27, 2008; accepted February 25, 2009.
Previously published online at DOI: 10.1373/clinchem.2007.100008
Mini-Review
renal androgens [dehydroepiandrostenedione (DHEA),5
DHEA-sulfate (DHEAS)], which in the adrenals are
produced in far higher amounts than androstenedione
and testosterone. Aromatase [cytochrome P (CYP)-
450 19, CYP19A1] adds 2 double bonds to ring A of
testosterone, yielding estradiol with an aromatic ring
A, and it also aromatizes ring A of androstenedione to
form estrone (E1).
One of the goals in the treatment of some breast
cancers is reduction of estrogen concentrations. This
can be achieved through the use of aromatase inhibi-
tors (AIs), which block the conversion of androgens to
estrogens (2 ). AIs do not sufficiently block estrogen
synthesis by the ovaries, however, but do block other
tissues from converting androgens to estrogens. For
this reason, AIs are used mostly in women who have
reached menopause, when the ovaries no longer syn-
thesize steroids. E1 synthesized mainly by aromatase
conversion of androstenedione is metabolized to sev-
eral steroid metabolites, including CYP1A2 conversion
to 2-hydroxyestrone (2-OHE1) and CYP3A4 and
CYP1A2 to 16-␣-hydroxyestrone (16-OHE1). The
former is weakly estrogenic and inhibits breast cell pro-
liferation (3– 6 ), whereas the latter is carcinogenic and
genotoxic, enhances breast cell growth, and increases
DNA synthesis and oncogene expression (3 ). Attempts
to modify the 2-OHE1:16-OHE1 ratio in young
women using the antidepressant fluoxetine are cur-
rently being explored (6 ). Fluoxetine is a known inhib-
itor of multiple P450 isoenzymes, including 3A4, 2C9,
and 2D6, and known to affect estrogen concentrations.
Methods of Measurement
Endogenous steroids have been measured by immuno-
assay (IA) (7–12 ), GC-MS (13–15 ), and high-
performance liquid chromatography–tandem mass
spectrometry (LC-MS/MS) (16 –21 ). An evaluation of
the results for steroid measurement in the College of
American Pathologists (CAP) proficiency testing (PT)
5
Nonstandard abbreviations: DHEA, dehydroepiandrostenedione; DHEAS, DHEA-
sulfate; CYP, cytochrome P; E1, estrone; AI, aromatase inhibitor; 2-OHE1,
2-hydroxyestrone; 16-OHE1, 16-␣-hydroxyestrone; IA, immunoassay; LC-MS/
MS, liquid chromatography–tandem mass spectrometry; CAP, College of Amer-
ican Pathologists; PT, proficiency testing; ESI, electrospray ionization; LOD, limit
of detection; APPI, atmospheric pressure photoionization; APCI, atmospheric
pressure chemical ionization; CAH, congenital adrenal hyperplasia; ACTH, ad-
renocorticotropic hormone.
1061
Mini-Review

Fig. 1. Steroid hormone synthetic pathways.

program is very informative. The majority of laborato- shown in Table 1 and is true for all steroids measured in
ries participating in this program employ IA methods, the CAP PT program. This contrasts with the good IA
each using a different antibody. For each method, CAP performance for the measurement of drugs such as
calculates the method mean, so it is possible to divide phenytoin, phenobarbital, carbamazepine, etc., where
the mean value of the method giving the highest mean the mean values for drugs in the CAP PT program for
by the mean value of the method giving the lowest
mean. Table 1 summarizes the IA results for one of the
CAP challenges for 2008. For this particular challenge, Table 1. Lack of specificity of steroid hormone
laboratories using the method giving the highest results immunoassays; data from CAP PT Program Y-A
differed from those using the procedure giving the low- Survey 2008.
est results by a factor of 2.8, 9.0, and 3.3 for testoster-
one, estradiol, and progesterone, respectively. Lowest Highest
This example illustrates vividly the magnitude of Analyte mean (L) mean (H) Factor H/L
the IA problem, due to many factors, including lack of Testosterone, ng/dL 52.6 148.7 2.8
specificity of antibodies purported to measure a partic- Estradiol, pg/mL 25.4 229.0 9.0
ular steroid. This poor IA performance for steroid mea- Progesterone, ng/mL 0.83 2.72 3.3
surement encompasses more than just the 3 steroids

1062 Clinical Chemistry 55:6 (2009)

Steroid Hormone Analysis by Tandem MS
Mini-Review

terone, and estradiol in 3 leading laboratories using

Table 2. MS/MS data of steroid hormones; CAP PT tandem mass spectrometry. Although excellent agree-
Program Y-A Survey 2008. ment was found for testosterone and progesterone, the
laboratories using derivatization at an extreme of pH
Lowest Highest for estradiol measurement had results approximately
Analyte value (L) value (H) Factor H/L
10%–20% higher than the laboratory that avoided der-
Testosterone, ng/dL ivatization. Derivatization methods are also lengthy
Testosterone no. 1 52 72 1.4 and therefore more time-consuming. Over the past 15
Testosterone no. 2 182 225 1.2 years, the detection limits with modern mass spec-
Estradiol, pg/mL trometers have improved greatly and have made the
Estradiol no. 1 109 109 1.0 derivatization of analytes unnecessary. For this reason,
Estradiol no. 2 628 630 1.0
we have been able to avoid derivatization approaches
for the measurement of steroids (17–19 ).
Progesterone, ng/mL
Progesterone no. 1 0.7 0.9 1.3
Type of ionization. Electrospray ionization (ESI) in the
Progesterone no. 2 8.1 8.6 1.1 negative mode yields the best results for the estrogens
(estradiol, estriol, E1, and 16-OHE1) (18 ). The method
used, which avoids derivatization, has a lower limit of
different IAs are almost identical. Stanczyk et al. (22 )
detection (LOD) of 1–2 pg/mL for all 4 estrogens when
also state that lack of standardization of steroid hor-
run on the API-5000 tandem mass spectrometer (Ap-
mone assays is a major deficiency in epidemiologic
plied Biosystems). Total sample requirement is only 0.2
studies. In postmenopausal women, the reported con-
mL, and total chromatography time for each estrogen
centrations of estradiol are highly variable, with nor-
profile is 8 min. The use of C-8 analytical columns (Su-
mal values differing by a factor of approximately 6
pelco LC-8-DB; 3.3 by 3.0 mm, 3 ␮m particle size) is
(22 ). This supports the CAP PT data reported above.
preferred over C-18 columns, as they markedly reduce
In contrast, Table 2 shows results for those labora-
retention times of the analytes in question. Our expe-
tories using MS/MS for steroid measurement. The
rience, and that of others (14, 18, 22 ), has shown that
high/low ratio is much better, ranging from 1.0 to 1.4.
IAs for estrogens have problems at low estrogen con-
It must be stated, however, that for testosterone, estra-
centrations (⬍80 pg/mL), frequently reporting falsely
diol, and progesterone the number of laboratories re-
increased results.
porting results by tandem mass spectrometry is small
For DHEA, DHEAS, androstenedione, testoster-
(9, 2, and 3, respectively, in this recent survey). This
one, progesterone, cortisol, 11-deoxycortisol, cortico-
could partially account for the apparently superior per-
sterone, and aldosterone, we have found that atmo-
formance of MS compared to IA. It also raises the ques-
spheric pressure photoionization (APPI) has potential
tion of whether all laboratories using MS/MS are com-
advantages over ESI or APCI (atmospheric pressure
plying with reporting their tandem mass spectrometry
chemical ionization) in that it is a soft ionization source
data in the CAP PT program.
that effectively ionizes these steroids fairly selectively,
leading to cleaner chromatograms. Alary (23 ) com-
FACTORS MERITING CONSIDERATION FOR MS/MS
pared APPI-MS/MS with APCI for the measurement of
MEASUREMENT OF STEROIDS
steroids in biological matrices and reported that the
Derivatization vs nonderivatization. This is currently an signal obtained using the APPI source was 3–10 times
important issue, with advocates on both sides. Deriva- more intense than that obtained employing the APCI
tization proponents claim that both enhanced limit of source. In our APPI method (19 ), the sample size is 0.2
quantification and specificity can be achieved by mL serum or plasma. After protein precipitation with
adopting this approach. This is questionable, however; acetonitrile containing the deuterated internal stan-
derivatization has its disadvantages, which include de- dards, the solution is vortex-mixed and centrifuged,
creased precision due to the added derivatization steps and the supernatant is injected directly onto a C-8 col-
(extraction, derivatization at an extreme of pH). Accu- umn, as with the estrogen profile assay. The column is
racy could be compromised as well through the possi- washed with buffer, the switching valve is activated,
ble hydrolysis of conjugates, which would clearly affect and steroids are eluted into the tandem mass spectrom-
the accuracy of the assay by giving falsely increased re- eter using a methanol gradient. The total chromatog-
sults. An example of such a problem could occur with raphy time for the steroid profile assay is 11 min. The
estradiol conjugates, which on hydrolysis would yield LOD varies from 1.5 to 10.0 pg/mL depending on the
estradiol. While serving on the CAP PT committee, we analyte (19 ). Whereas ion suppression has limited
compared the values obtained for testosterone, proges- the usefulness of many MS/MS methods, it is a rare

Clinical Chemistry 55:6 (2009) 1063

Mini-Review
occurrence with the method described. Use of deuter-
ated internal standards and an online sample wash step
are partly responsible for the good performance.
Profile vs single steroid testing. Large commercial refer-
ence laboratories have high daily volumes for many of
the clinically important steroids discussed above. To
accommodate this volume and meet the need for rapid
turnaround time with short chromatography time,
these laboratories have adopted a 1 tandem/1 steroid
philosophy, with a different tandem mass spectrometer
used for each steroid. Smaller teaching hospital labora-
tories with a less pressing workload can, on the other
hand, evaluate the potential role of steroid profile test-
ing. As LC-MS/MS allows the simultaneous measure-
ment of several steroids, sample sizes can be reduced
compared to IA platforms that require an additional
sample for each steroid measured; this is particularly
germane in the evaluation of infants, where specimen
size is limited.
Multiple circumstances in which steroid profiles have
been employed.
1. Congenital adrenal hyperplasia (CAH) is an inborn
error of steroid biosynthesis. CAH is a group of inher-
ited diseases caused by defective activity of 1 of 5 en-
zymes in the adrenal cortex. The defective enzyme
leads to decreased production of cortisol (causing an
increased corticotropin) and excess production of hor-
mones proximal to the defect. The 2 most common
forms of CAH are caused by either 21-hydroxylase de-
ficiency (defect in the P450c21 enzyme) or 11-␤-
hydroxylase deficiency (defect in P450c11 enzyme). In-
dividuals with CAH due to 11- or 21-hydroxylase
enzyme deficiency cannot produce adequate amounts
of cortisol and, in some cases, are also aldosterone de-
ficient. These hormones are essential in glucose metab-
olism and sodium reabsorption. Untreated CAH can
lead to sudden adrenal insufficiency, with dehydration,
shock, and even death. Steroids that have been recom-
mended for the assessment of CAH are cortisol, andro-
stenedione, and 17-hydroxyprogesterone (24 –26 ). At
Children’s National Medical Center, we routinely use a
broad 11-steroid profile to improve the specificity of
screening for CAH caused by either 21-hydroxylase or
11-hydroxylase deficiencies (19 ).
Routine newborn screening for congenital adrenal
hyperplasia suffers from a high rate of false-positive
and false-negative results when using an IA to measure
17-OH progesterone concentrations, especially in crit-
ically ill newborns and preterm neonates. Therefore,
immediate reanalysis of all IA results above the cutoff
using LC-MS/MS can allow a clear distinction of af-
fected and nonaffected newborns (24, 26 ).
1064 Clinical Chemistry 55:6 (2009)
2. The evaluation of adrenal insufficiency is histori-
cally recommended by measuring cortisol at 0, 30, and
60 min after an adrenocorticotropic hormone (ACTH)
stimulation test (27–29 ). We have improved the diag-
nostic reliability of this approach by measuring the 3
steroids aldosterone, cortisol, and most importantly,
11-deoxycortisol at 0, 30, and 60 min. Including aldo-
sterone in the profile allows the differentiation of pri-
mary from secondary adrenal insufficiency. In primary
adrenal insufficiency, no aldosterone response is ob-
served, whereas an adequate response is found in sec-
ondary adrenal insufficiency (30 ). The concentration
of 11-deoxycortisol increases 15- to 20-fold in controls
after an ACTH stimulation test, which compares to an
approximately 3-fold increase for the more tradition-
ally measured analyte cortisol. Our study demon-
strated greater diagnostic accuracy if these 3 steroids
were measured instead of measuring only cortisol (30 ).
3. We have also assessed the role of steroid profiles in
patients with chronic prostatitis/chronic pelvic pain syn-
drome. Our results suggest reduced activity of CYP21A2
(P450c21), which is the 21-hydroxylase enzyme that con-
verts progesterone to 11-desoxycorticosterone and 17-
hydroxyprogesterone to 11-deoxycortisol (31 ) (Fig. 1).
4. We assessed whether steroid profiles provided in-
sight into the reasons for premature adrenarche and
infants with genital hair. In both these groups, the con-
centrations of testosterone, androstenedione, DHEA,
and DHEAS were somewhat higher than in age-
matched controls (32 ). We have also assessed the ref-
erence intervals for these steroids during pregnancy
and 1 year postpartum using isotope dilution tandem
mass spectrometry (33 ).
5. Sera from active smokers, passive smokers, and
nonsmokers have been analyzed for 15 steroid hor-
mones and thyroid hormones to examine the associa-
tions between smoke exposure and hormone concen-
trations (34, 35 ). Although we do not know whether
the blood concentrations of the hormones reflect
changes that parallel physiological variation in steroid
hormone concentrations, the assumption is that
differences reflect associations with tobacco smoke
exposure.
6. LC-MS/MS after solid-phase extraction has been
used in lipidomic profiling of some female steroid hor-
mones in human urine, and can be potentially applied
clinically and to metabolomic research (36 ).
7. Diabetes strongly affects neuroactive steroids in the
nervous system. LC-MS/MS assessment of the concen-
trations of neuroactive steroids provides a basis for new
therapeutic tools based on neuroactive steroids aimed
at counteracting diabetic neuropathy (37 ).
8. Even in trace amounts, estrogens such as E2, E1,
estriol, and 17␣-ethinyl estradiol may have adverse ef-
fects on humans and the aquatic ecosystem. It is there-
Steroid Hormone Analysis by Tandem MS
Mini-Review

fore essential to be able to reliably determine trace
amounts (at environmentally relevant concentrations)
of steroid estrogens in water. Using LC-MS/MS, it is
now possible to detect these chemicals in small samples
of water at concentrations as low as 0.04 ng/L (38 – 41 ).
9. Finally, steroid profiling has been used to assess
changes in adrenal steroids before and after a 56-km
ultramarathon race (42 ). Concentrations of the min-
eralocorticoids corticosterone and aldosterone in-
creased significantly, as did concentrations of the glu-
cocorticoids cortisol, 11-deoxycortisol, and 17-OH
progesterone and the adrenal steroids DHEA, DHEAS,
and androstenedione (P ⬍ 0.0001 for all).
Some future areas for research include the assess-
ment of the role of neurosteroids in epilepsy, particu-
larly in pubertal girls in whom a marked increase in
seizure activity has been found (43– 45 ), and analysis of
androgens in males, patients with benign prostatic hy-
perplasia, and prostate cancer (46, 47 ).
In conclusion, LC-MS/MS now affords the speci-
ficity, imprecision, and limits of quantification neces-
sary for the reliable measurement of steroids in human
fluids, thereby enhancing diagnostic capabilities, par-
ticularly when steroid profiles are available. Major ad-
vantages of tandem mass spectrometry include small
sample size, the simultaneous measurement of many
analytes, and enhanced specificity compared to IA
methods. Mass spectrometric methods are still fairly
labor intensive, and certainly require a higher level of
laboratory expertise than do IA platforms. Occasional
interferences when using mass spectrometric methods
have been described, such as prednisolone/prednisone
metabolite interference in urinary free cortisol mea-
surements (48 ). It should be noted that currently reim-
bursement for steroid profile testing is not yet ap-
proved by Medicare (with the exception of the CAH
steroid profile), nor are steroid profiles ordered fre-
quently by clinicians. This could well change as steroid
hormone profiling becomes more appreciated in the
years ahead. Although mass spectrometric assays are
not always more precise than IAs, they are more spe-
cific for measuring the analyte of interest. By omitting
extraction and derivatization steps, the steroid tandem
mass spectrometric procedures described here have
good intrarun and interrun imprecision (18, 19 ). Drug
interference has been tested and found not to be a
problem for the steroid and estrogen profiles reported
in this review (18, 19 ). These two methods are also rel-
atively free of ion suppression.
Author Contributions: All authors confirmed they have contributed to
the intellectual content of this paper and have met the following 3 re-
quirements: (a) significant contributions to the conception and design,
acquisition of data, or analysis and interpretation of data; (b) drafting
or revising the article for intellectual content; and (c) final approval of
the published article.
Authors’ Disclosures of Potential Conflicts of Interest: Upon
manuscript submission, all authors completed the Disclosures of Poten-
tial Conflict of Interest form. Potential conflicts of interest:
Employment or Leadership: None declared.
Consultant or Advisory Role: S.J. Soldin, NIH, and NMS.
Stock Ownership: None declared.
Honoraria: S.J. Soldin, AACC, CSCC, and CAMB.
Research Funding: S.J. Soldin, NMS, Applied Biosystems, and NIH.
Expert Testimony: None declared.
Role of Sponsor: The funding organizations played no role in the
design of study, choice of enrolled patients, review and interpretation
of data, or preparation or approval of manuscript.

1. Holst JP, Soldin OP, Guo T, Soldin SJ. Steroid
hormones: relevance and measurement in the clin-
ical laboratory. Clin Lab Med 2004;24:105–18.
2. Swain SM. Aromatase inhibitors: a triumph of
translational oncology. N Engl J Med 2005;353:
2807–9.
3. Bradlow HL, Davis DL, Lin G, Sepkovic D, Tiwari
R. Effects of pesticides on the ratio of 16 alpha/
2-hydroxyestrone: a biologic marker of breast
cancer risk. Environ Health Perspect 1995;103
Suppl 7:147–50.
4. Chen Z, Zheng W, Dunning LM, Anderson KG,
Parrish RS, Holtzman JL. Within-person variability
of the ratios of urinary 2-hydroxyestrone to
16alpha-hydroxyestrone in Caucasian women.
Steroids 1999;64:856 –9.
5. Shou M, Korzekwa KR, Brooks EN, Krausz KW,
Gonzalez FJ, Gelboin HV. Role of human hepatic
cytochrome P450 1A2 and 3A4 in the metabolic
activation of estrone. Carcinogenesis 1997;18:
207–14.
6. Thompson DS, Kirshner MA, Klug TL, Kastango
References
KB, Pollock BG. A preliminary study of the effect
of fluoxetine treatment on the 2:16-alpha-
hydroxyestrone ratio in young women. Ther Drug
Monit 2003;25:125– 8.
7. Carter GD, Carter R, Jones J, Berry J. How accu-
rate are assays for 25-hydroxyvitamin D? Data
from the international vitamin D external quality
assessment scheme. Clin Chem 2004;50:2195–7.
8. Dorgan JF, Fears TR, McMahon RP, Aronson
Friedman L, Patterson BH, Greenhut SF. Measure-
ment of steroid sex hormones in serum: a com-
parison of radioimmunoassay and mass spec-
trometry. Steroids 2002;67:151– 8.
9. Hollis BW, Kamerud JQ, Kurkowski A, Beaulieu J,
Napoli JL. Quantification of circulating 1,25-
dihydroxyvitamin D by radioimmunoassay with
125
I-labeled tracer. Clin Chem 1996;42:586 –92.
10. Hsing AW, Stanczyk FZ, Belanger A, Schroeder P,
Chang L, Falk RT, et al. Reproducibility of serum
sex steroid assays in men by RIA and mass spec-
trometry. Cancer Epidemiol Biomarkers Prev
2007;16:1004 – 8.
11. Poulson K, Sancho J, Haber E. A simplified radio-
immunoassay for plasma aldosterone employing
an antibody of unique specificity. Clin Immunol
1974;2:373– 80.
12. Schioler V, Thode J. Six direct radioimmunoassays of
estradiol evaluated. Clin Chem 1988;34:949 –52.
13. Andrew R. Clinical measurement of steroid me-
tabolism. Best Pract Res Clin Endocrinol Metab
2001;15:1–16.
14. Santen RJ, Demers L, Ohorodnik S, Settlage J,
Langecker P, Blanchett D, et al. Superiority of gas
chromatography/tandem mass spectrometry as-
say (GC/MS/MS) for estradiol for monitoring of
aromatase inhibitor therapy. Steroids 2007;72:
666 –71.
15. Wolthers BG, Kraan GP. Clinical applications of
gas chromatography and gas chromatography-
mass spectrometry of steroids. J Chromatogr A
1999;843:247–74.
16. Draisci R, Palleschi L, Ferretti E, Lucentini L, Cam-
marata P. Quantitation of anabolic hormones and
their metabolites in bovine serum and urine by

Clinical Chemistry 55:6 (2009) 1065

Mini-Review
liquid chromatography-tandem mass spectrome-
try. J Chromatogr A 2000;870:511–22.
17. Guo T, Chan M, Soldin SJ. Steroid profiles using
liquid chromatography-tandem mass spectrome-
try with atmospheric pressure photoionization
source. Arch Pathol Lab Med 2004;128:469 –75.
18. Guo T, Gu J, Soldin OP, Singh RJ, Soldin SJ. Rapid
measurement of estrogens and their metabolites
in human serum by liquid chromatography-
tandem mass spectrometry without derivatiza-
tion. Clin Biochem 2008;41:736 – 41.
19. Guo T, Taylor RL, Singh RJ, Soldin SJ. Simulta-
neous determination of 12 steroids by isotope
dilution liquid chromatography-photospray ion-
ization tandem mass spectrometry. Clin Chim
Acta 2006;372:76 – 82.
20. Nelson RE, Grebe SK, OKane DJ, Singh RJ. Liquid
chromatography-tandem mass spectrometry as-
say for simultaneous measurement of estradiol
and estrone in human plasma. Clin Chem 2004;
50:373– 84.
21. Xu X, Roman JM, Issaq HJ, Keefer LK, Veenstra TD,
Ziegler RG. Quantitative measurement of endoge-
nous estrogens and estrogen metabolites in human
serum by liquid chromatography-tandem mass
spectrometry. Anal Chem 2007;79:7813–21.
22. Stanczyk FZ, Lee JS, Santen RJ. Standardization of
steroid hormone assays: why, how, and when?
Cancer Epidemiol Biomarkers Prev 2007;16:
1713–9.
23. Alary JF. Comparative study: LC/MS/MS analysis
of four steroid compounds using a new photo-
ionization source and a conventional APCI
source. In: Proceedings of the 49th ASMS Con-
ference on Mass Spectrometry and Allied Topics;
2001 May 27–31; Chicago. [Santa Fe (NM)]:
American Society for Mass Spectrometry; 2001.
Abstract nr A010942.
24. Lacey JM, Minutti CZ, Magera MJ, Tauscher AL,
Casetta B, McCann M, et al. Improved specificity
of newborn screening for congenital adrenal hy-
perplasia by second-tier steroid profiling using
tandem mass spectrometry. Clin Chem 2004;50:
621–5.
25. Minutti CZ, Lacey JM, Magera MJ, Hahn SH,
McCann M, Schulze A, et al. Steroid profiling by
tandem mass spectrometry improves the positive
predictive value of newborn screening for con-
genital adrenal hyperplasia. J Clin Endocrinol
Metab 2004;89:3687–93.
26. Janzen N, Peter M, Sander S, Steuerwald U, Ter-
hardt M, Holtkamp U, et al. Newborn screening
for congenital adrenal hyperplasia: additional ste-
roid profile using liquid chromatography-tandem
mass spectrometry. J Clin Endocrinol Metab
2007;92:2581–9.
27. Crowley S, Hindmarsh PC, Honour JW, Brook CG.
1066 Clinical Chemistry 55:6 (2009)
Reproducibility of the cortisol response to stimu-
lation with a low dose of ACTH(1–24): the effect
of basal cortisol levels and comparison of low-
dose with high-dose secretory dynamics. J Endo-
crinol 1993;136:167–72.
28. Grinspoon SK, Biller BM. Clinical review 62: lab-
oratory assessment of adrenal insufficiency. J Clin
Endocrinol Metab 1994;79:923–31.
29. May ME, Carey RM. Rapid adrenocorticotropic
hormone test in practice: retrospective review.
Am J Med 1985;79:679 – 84.
30. Holst JP, Soldin SJ, Tractenberg RE, Guo T, Kun-
dra P, Verbalis JG, et al. Use of steroid profiles in
determining the cause of adrenal insufficiency.
Steroids 2007;72:71– 84.
31. Dimitrakov J, Joffe HV, Soldin SJ, Bolus R, Buff-
ington CA, Nickel JC. Adrenocortical hormone
abnormalities in men with chronic prostatitis/
chronic pelvic pain syndrome. Urology 2008;71:
261– 6.
32. Kaplowitz P, Soldin SJ. Steroid profiles in serum
by liquid chromatography-tandem mass spec-
trometry in infants with genital hair. J Pediatr
Endocrinol Metab 2007;20:597– 605.
33. Soldin OP, Guo T, Weiderpass E, Tractenberg RE,
Hilakivi-Clarke L, Soldin SJ. Steroid hormone lev-
els in pregnancy and 1 year postpartum using
isotope dilution tandem mass spectrometry. Fertil
Steril 2005;84:701–10.
34. Soldin OP, Loffredo CA, Shields PG, Marion C,
Ressom H, Landy H. Hormone changes in women
of reproductive age associations with genetics
and tobacco smoke exposure. From: Flight Atten-
dant Medical Research Institute’s Sixth Scientific
Symposium; 2007 May 14 –16; Miami, Fla.
35. Soldin OP, Soldin SJ, Ressom H, Landy HJ. Hor-
mone changes in women of reproductive age
associated with tobacco smoke exposure using
metabolomics. Soc Gynecol Invest Annu Meeting,
2008; San Diego, CA.
36. Alvarez Sanchez B, Capote FP, Jimenez JR, Luque de
Castro MD. Automated solid-phase extraction for
concentration and clean-up of female steroid hor-
mones prior to liquid chromatography-electrospray
ionization-tandem mass spectrometry: an approach
to lipidomics. J Chromatogr A 2008;1207:46 –54.
37. Caruso D, Scurati S, Maschi O, De Angelis L,
Roglio I, Giatti S, et al. Evaluation of neuroactive
steroid levels by liquid chromatography-tandem
mass spectrometry in central and peripheral ner-
vous system: effect of diabetes. Neurochem Int
2008;52:560 – 8.
38. Lin YH, Chen CY, Wang GS. Analysis of steroid
estrogens in water using liquid chromatography/
tandem mass spectrometry with chemical derivat-
izations. Rapid Commun Mass Spectrom 2007;21:
1973– 83.
39. Reddy S, Iden CR, Brownawell BJ. Analysis of
steroid conjugates in sewage influent and efflu-
ent by liquid chromatography-tandem mass spec-
trometry. Anal Chem 2005;77:7032– 8.
40. Schlusener MP, Bester K. Determination of steroid
hormones, hormone conjugates and macrolide
antibiotics in influents and effluents of sewage
treatment plants utilising high-performance liquid
chromatography/tandem mass spectrometry with
electrospray and atmospheric pressure chemical
ionisation. Rapid Commun Mass Spectrom 2005;
19:3269 –78.
41. Stanford BD, Weinberg HS. Isotope dilution for
quantitation of steroid estrogens and nonylphe-
nols by gas chromatography with tandem mass
spectrometry in septic, soil, and groundwater ma-
trices. J Chromatogr A 2007;1176:26 –36.
42. Hew-Butler T, Jordaan E, Stuempfle KJ, Speedy
DB, Siegel AJ, Noakes TD, et al. Osmotic and
nonasmotic regulation of arginine vasopressin
during prolonged endurance exercise. J Clin En-
docrinol Metab 2008;93:2072– 8.
43. Higashi T, Nagahama A, Otomi N, Shimada K.
Studies on neurosteroids XIX. Development and
validation of liquid chromatography-tandem
mass spectrometric method for determination of
5alpha-reduced pregnane-type neurosteroids in
rat brain and serum. J Chromatogr 2007;848:
188 –99.
44. Hill M, Bicikova M, Parizek A, Havlikova H, Klak
J, Fajt T, et al. Neuroactive steroids, their precur-
sors and polar conjugates during parturition and
postpartum in maternal blood: 2. Time profiles of
pregnanolone isomers. J Steroid Biochem Mol
Biol 2001;78:51–7.
45. Hill M, Parizek A, Bicikova M, Havlikova H, Klak
J, Fait T, et al. Neuroactive steroids, their precur-
sors, and polar conjugates during parturition and
postpartum in maternal and umbilical blood: 1.
Identification and simultaneous determination of
pregnanolone isomers. J Steroid Biochem Mol
Biol 2000;75:237– 44.
46. Higashi T, Yamauchi A, Shimada K, Koh E,
Mizokami A, Namiki M. Determination of pros-
tatic androgens in 10 mg of tissue using liquid
chromatography-tandem mass spectrometry with
charged derivatization. Anal Bioanal Chem 2005;
382:1035– 43.
47. Zhao M, Baker SD, Yan X, Zhao Y, Wright WW,
Zirkin BR, et al. Simultaneous determination of
steroid composition of human testicular fluid us-
ing liquid chromatography tandem mass spec-
trometry. Steroids 2004;69:721– 6.
48. Kushnir MM, Rockwood AL, Nelson GJ, Terry AH,
Meikle AW. Liquid chromatography tandem mass
spectrometry analysis of urinary free cortisol. Clin
Chem 2003;49:965–7.